During hypoosmotic stress, membrane vesicles enriched in acetylcholine receptors become more permeable to external tracer molecules. When vesicles are immersed in 3 volumes of water containing 22Na+, 50-70% of 22Na+ equilibration is attained within 90 s. On the other hand, the uptake of 22 Na+ is greatly diminished only 6-10 s after an osmotic shock, and vesicle resealing is completed within 15 s. Furthermore, 90 s after osmotic shock, efflux rates are comparable to those of native vesicles, which also indicates that the vesicles have resealed. During osmotic shock, the entry of molecules into the vesicles increases with the strength of the osmotic shock and also depends on the size of the permeant. With a given strength of osmotic shock, the large molecule [3H]inulin (Mr 5000) is taken up less than the smaller molecules 22Na+ and [3H]sucrose. In addition, alpha-bungarotoxin binding latency of the vesicles is not affected by osmotic shock, indicating that the sideness of the vesicles remains unchanged. The acetylcholine receptors in the vesicles remain functional after osmotic shock. For example, 90 s after 22Na+ and [3H]sucrose are loaded into vesicles by osmotic shock, only 22Na+ is released by dilution in a buffer containing carbamoylcholine (carbamylcholine). Also, the influx of 22 Na+ into previously shocked vesicles can be specifically stimulated by carbamoylcholine. Such stimulations in the shocked vesicles can be blocked by d-tubocurarine or alpha-bungarotoxin, and they can be desensitized by preincubation with carbamoylcholine. These results suggest the possibility of using osmotic shock to load molecular probes into these membrane vesicles, which could provide a powerful tool for studying inner surfaces of the intact vesicles.